pISSN 1738-2696 · eISSN 2212-5469 http://dx.doi.org/10.1007/s13770-015-9066-x
ORIGINAL ARTICLE
Extracellular Calcium-Binding Peptide-Modified Ceramics Stimulate Regeneration of Calvarial Bone Defects Ju Ang Kim1†, Young-Ae Choi1†, Hui-Suk Yun2, Yong Chul Bae3, Hong-In Shin1, Eui Kyun Park1* Department of Oral Pathology and Regenerative Medicine, School of Dentistry, IHBR, Kyungpook National University, Daegu, Korea Powder and Ceramics Division, Korea Institute of Materials Science (KIMS), Changwon, Korea 3 Department of Oral Anatomy and Neurobiology, School of Dentistry, Kyungpook National University, Daegu, Korea 1 2
Secreted protein, acidic, cysteine-rich (SPARC)-related modular calcium binding 1 (SMOC1) has been implicated in the regulation of osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs). In this study, we found that a peptide (16 amino acids in length), which is located in the extracellular calcium (EC) binding domain of SMOC1, stimulated osteogenic differentiation of human BMSCs in vitro and calvarial bone regeneration in vivo. Treatment of BMSCs with SMOC1-EC peptide significantly stimulated their mineralization in a dose-dependent manner without changing their rate of proliferation. The expression of osteogenic differentiation marker genes, including type 1 collagen and osteocalcin, also increased in a dose-dependent manner. To examine the effect of the SMOC1-EC peptide on bone formation in vivo, the peptide was covalently immobilized onto hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) particles. X-ray photoelectron spectroscopy analysis showed that the peptide was successfully immobilized onto the surface of HA/β-TCP. Implantation of the SMOC1-EC peptide-immobilized HA/β-TCP particles into mouse calvarial defects and subsequent analyses using microcomputed tomography and histology showed significant bone regeneration compared with that of calvarial defects implanted with unmodified HA/β-TCP particles. Collectively, our data suggest that a peptide derived from the EC domain of SMOC1 induces osteogenic differentiation of human BMSCs in vitro and efficiently enhances bone regeneration in vivo. Tissue Eng Regen Med 2016;13(1):57-65 Key Words: Secreted protein, acidic, cysteine-rich-related modular calcium binding 1; Extracellular calcium domain; Peptide; Hydroxyapatite/β-tricalcium phosphate; Osteogenesis; bone marrow mesenchymal stem cells
INTRODUCTION Bioactive peptides are defined as particular protein fragments derived from either natural or artificial proteins [1]. They have been implicated in regulating a variety of biological functions to exert antioxidant [2], antimicrobial [3], antihypertensive [4], antithrombotic [5], and immunomodulatory activities [6]. Bioactive peptides can also mimic the biological activities of proteinaceous growth factors. In particular, peptides derived from bone morphogenetic proteins (BMPs) have activities similar to those of full-length BMPs and can stimulate bone formaReceived: July 29, 2015 Revised: August 24, 2015 Accepted: August 27, 2015 *Corresponding author: Eui Kyun Park, Department of Oral Pathology and Regenerative Medicine, School of Dentistry, IHBR, Kyungpook National University, 2177 Dalgubeol-daero, Jung-gu, Daegu 41940, Korea. Tel: 82-53-420-4995, Fax: 82-53-428-4995, E-mail:
[email protected] These authors contributed equally to this work.
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tion [7]. Although BMPs have been applied for the therapeutic treatment of bone defects or diseases [8], their clinical application requires careful consideration because they may cause severe adverse effects such as organogenesis, apoptosis, tumorigenicity, and immunogenicity [9,10]. In addition, the application of concentrated BMPs without immobilization occasionally leads to ectopic bone formation [10], and their long-term effects and release patterns are still unknown. To overcome these issues, the controlled release of BMPs after their conjugation to materials is necessary. In the conjugation process, however, peptides have the advantage of better stability compared with that of native proteins. Moreover, peptides can be massively and economically synthesized in laboratories. Biomaterials modified with growth factor-derived biomimetic peptides can stimulate cellular healing or regeneration, and thus can be used to enhance the performance of scaffolds for hard tissue engineering [11].
© The Korean Tissue Engineering and Regenerative Medicine Society
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Kim et al. Bone Regeneration by SMOC1-EC Peptide-Immobilized HA/β-TCP
For the regeneration of hard tissue, a variety of biomaterials has been developed, including bioactive glasses, ceramics, and other composites. In particular, hydroxyapatite-based ceramics have been widely used for orthopedic and dental surgeries because they have osteoconductive properties [12]. Recently, hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) composite ceramics have been broadly used for the regeneration of bone defects because of their degradability, biocompatibility, and osteoconductivity [13]. However, their biological performance needs to be improved to enhance bone regeneration. An approach that may achieve such an improvement in performance is the immobilization of bioactive molecules onto the HA/β-TCP composite ceramics. Previous reports have shown that surface modification of ceramics with bioactive peptides resulted in enhanced bone regeneration [14,15]. Previously, we found that SPARC-related modular calcium binding 1 (SMOC1) plays an important role in regulating the osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs) [16]. SMOC1 was first identified as a member of the SPARC (BM-40 or osteonectin) family and is a calcium-binding protein in the basement membrane [17]. It binds to several extracellular matrix (ECM) proteins and plays roles in bone remodeling and cancer metastasis [18]. SMOC1 was reported to be downregulated by cytokines and nitric oxide in rat mesangial cells [19]. On the other hand, SMOC1 mutations in humans are known to cause a genetic disorder called the Waardenburg Anophthalmia Syndrome, which results in abnormalities of the limbs and eyes [20]. SMOC1 has distinct domains, including 1 follistatin-like domain, 2 thyroglobulinlike domains, 1 extracellular calcium (EC) domain, and 1 SMOC1 unique domain [21]. The EC domain has calcium-binding sites, and calcium ions are critical for osteogenic differentiation and mineralization. We selected peptide sequences from each domain of SMOC1, and we found that a peptide fragment derived from the EC domain of SMOC1 stimulated osteogenic differentiation of human BMSCs in vitro and bone regeneration in vivo.
MATERIALS AND METHODS Materials
TriOSiteTM was purchased from Zimmer (Vigneux-de-Bretagne, France) to generate SMOC1-EC peptide-immobilized HA/β-TCP particles. For peptide immobilization, HA/β-TCP granules were ground with a mortar, and sieved to a size of 100– 200 μm. Poly (ethylene glycol disuccinimidyl succinate) [PEG(SS)2] and 3-aminopropyl-triethoxysilane (APTES) were obtained from SunBio (Anyang, Korea) and Sigma-Aldrich (Milwaukee, WI, USA), respectively. The amino acid sequence of the SMOC1-EC peptide was selected from the region of the
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second α-helix of the EF-hand calcium-binding motif within the EC domain. The SMOC1-EC peptide was optimized for net charge and solubility using Peptide Property Calculator (http://www.basic.northwestern.edu/biotools/proteincalc. html). The SMOC1-EC peptide consists of 16 amino acids covering residues 399–414 in the human SMOC1 peptide sequence. The isoelectric point of the peptide was 8.470 and its average hydrophilicity was 0.92. The percentage of hydrophilic residues among total residues was 56.25%. The molecular weight of the peptide was 1976.45 Da, and its net charge at pH 7.0 was 1.9. The SMOC1-EC peptide (KCARRFTDYCDLNKDK) was synthesized with 95% purity by Peptron (Daejeon, Korea).
Immobilization of the SMOC1-EC peptide onto the surface of HA/β-TCP particles
The surface immobilization procedure was described previously [14]. SMOC1-EC peptide was immobilized to HA/β-TCP particles (100–200 μm) using 3 steps. First, the free hydroxyl groups of the HA/β-TCP particles were silanized by treatment with the primary amine APTES (500 mM) in anhydrous ethanol for 6 h at 25°C. Second, the silanized HA/β-TCP particles were washed with ethanol 5 times and then reacted with 10 mM of PEG-(SS)2 in 2:3 dimethylformamide and ethanol solution for another 6 h. Third, the PEGylated particles were rinsed with ethanol and phosphate buffered saline and then reacted with 10 μM of SMOC1-EC peptide with gentle rotation for 12 h at 4°C. The processed HA/β-TCP was subsequently cleaned with ethanol and freeze-dried before storage.
Validation of the immobilization of SMOC1-EC peptide onto HA/β-TCP
Dried PEGlyated and SMOC1-EC peptide-immobilized HA/ β-TCP particles were analyzed using X-ray photoelectron spectroscopy (XPS) (Quantera SXM, ULVAC-PHI, Japan). The XPS spectra were obtained using algorithms of a magnesium anode at 13 kV and 30 mA. Further analyses of XPS and XPS-C1s were followed as described earlier [14]. The XPS-C1s band spectra were separated into multi-peak spectra using ORIGIN 6.0 software (OriginLab Corp., Northampton, MA, USA).
Osteogenic differentiation and cytotoxicity of human BMSCs treated with SMOC1-EC peptide
To examine the effect of the synthetic SMOC1-EC peptide on osteogenic differentiation in vitro, human BMSCs were seeded at a density of 1.3×104 cells per cm2 on a culture plate (Falcon, San Jose, CA, USA), in triplicate. After 24 h, they were treated with osteogenic induction medium [α-MEM with 10% (v/v) fetal bovine serum, 1% (v/v) antibiotics solution, 50 μg/mL ascorbic acid, 10 nM dexamethasone, and 10 mM β-glycerophos-
phate] containing SMOC1-EC peptides (0, 0.1, 0.5, or 2.5 μM). After 21 days, the cells were stained with Alizarin red S. The mineral depositions were visualized with a light microscope (Olympus, Tokyo, Japan), and dye extracted using 10% (w/v) cetylpyridinium chloride was measured at OD570 as described previously [22]. To analyze the effect of the SMOC1-EC peptide on the expression of osteogenic differentiation marker genes, total ribonucleic acid was isolated using TRI-SolutionTM (Bio Science Korea, Gyeongsan, Korea), and complementary DNA was synthesized using SuperScriptTM II (Invitrogen, Carlsbad, CA, USA). The subsequent thermal cycling was analyzed using a C1000TM thermal cycler (Bio-Rad, Foster City, CA, USA). The osteogenic differentiation marker genes included osteonectin (ON), osteocalcin (OC), and type 1 collagen (COL1). The primer sequences of ON, OC, and COL1 were described previously [16]. Each sample was analyzed in triplicate and normalized to GAPDH expression. To examine the cytotoxicity of the SMOC1EC peptide, 700 human BMSCs were seeded in each well of a 96-well plate. The cells were treated with varying concentrations (0, 0.1, 0.5, and 2.5 μM) of SMOC1-EC peptide, and cytotoxicity assessment was performed using the methylthiazol tetrazolium (MTT) assay (Sigma-Aldrich) as described previously [23].
Animal study
Animal experiments were performed under the guidance of the Institutional Ethics Committee of Kyungpook National University. Healthy Institute for Cancer Research (ICR) mice (male, 6 weeks old) were used for the experiment, and anesthetization was performed as previously described [24]. The head was shaved and cleaned up with povidone-iodine solution and the parietal skin and periosteum were incised. The calvarial defect was generated by trephine burr (4 mm in diameter) with a dental
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To analyze the regeneration of the calvarial defects, the fixed calvariae were scanned using an X-eye micro-CT system (SEC, Seoul, Korea) with an anode current of 80 μA and an anode voltage of 72 kV. The imaging data were achieved using half-scan CT algorithms, and scanned planes of 512×512 were reconstructed using the HARMONY program (DRGEM, Seoul, Korea). Subsequently, 3-dimensional (3D) images were created using the iCAT3D program (Mevisys, Daejeon, Korea). Reconstructed 3D images were further analyzed using CTAn (Bruker microCT, Kontich, Belgium) to calculate the bone volume as a proportion of tissue volume (%).
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hand piece (Marathon, Seoul, Korea). SMOC1-EC peptide-immobilized HA/β-TCP particles (10 mg) were mixed with fibrin glue (Greenplast kit VR, Green Cross, Seoul, Korea) [25] and molded into a flattened disc of 4 mm diameter and 1 mm thickness. Then, the disc-shaped particles were implanted into the defect region of the calvariae. Animals were implanted with either unmodified HA/β-TCP (n=5) or SMOC1-EC-immobilized HA/ β-TCP (n=5). Six weeks after implantation, the mice were sacrificed and the calvariae were isolated and fixed with 4% (w/v) formaldehyde in phosphate buffered saline (pH 7.4) for 48 h. The specimens were decalcified with 0.5 M ethylenediaminetetraacetic acid (pH 8.0) for 7 days and then processed for paraffin embedding. The paraffin blocks were sectioned at a thickness of 5 μm using a microtome (Leica Microsystems, Nussloch, Germany), and sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome. The new bone area was measured and analyzed using IMT i-Solution software (IMT Technology, Daejeon, Korea).
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Figure 1. Cytotoxicity of SMOC1-EC peptide in human BMSCs. Human BMSCs were seeded in 96 well plates and treated with increasing doses of SMOC1-EC peptides for 1 week. At the indicated time point, MTT assay was performed (n=3). There are no statistical differences. SMOC1-EC: SPARC-related modular calcium binding 1-extracellular calcium, BMSCs: bone marrow mesenchymal stem cells, MTT: methylthiazol tetrazolium. www.term.or.kr 59
Kim et al. Bone Regeneration by SMOC1-EC Peptide-Immobilized HA/β-TCP
Statistical analysis
Statistical analysis was performed with SPSS 21.0 (one-way analysis of variance) and p values of less than 0.05, 0.02, or 0.01 were considered significant.
RESULTS Cytotoxicity of SMOC1-EC peptide in human BMSCs
The cytotoxicity of the SMOC1-EC peptide was examined using the MTT assay. Human BMSCs isolated from 2 independent donors were treated with SMOC1-EC peptide at the concentrations of 0, 0.1, 0.5, and 2.5 μM, and MTT assay was performed after 1, 3, 5, and 7 days of culture. The results showed that the SMOC1-EC peptide did not significantly affect the proliferation of BMSCs derived from donor 1 or donor 2, compared with the control (Fig. 1). These results suggest that the SMOC1-EC peptide did not significantly exert cytotoxicity in human BMSCs.
Effect of SMOC1-EC peptide on mineralization and osteogenic differentiation of human BMSCs
To examine the effect of the SMOC1-EC peptide on the osteogenic differentiation of BMSCs, human BMSCs were cultured in osteogenic induction media with or without the indicated concentrations of the SMOC1-EC peptide for 3 weeks and mineralization was evaluated by Alizarin red S staining. The results showed that the SMOC1-EC peptide significantly increased calcium deposition in human BMSCs in a concentration-dependent manner (Fig. 2A, left). Quantification of the extracted dye confirmed an increase in calcium deposition by the SMOC1-EC peptide compared with the control (p
